Insulin-related lipohypertrophy: ultrasound characteristics, risk factors, and impact of glucose fluctuations

Lipohypertrophy (LHT) has been suggested as an outcome of the adipogenic effects of insulin injection-related tissue trauma. It commonly occurs in the clinical setting, but the current understanding of LHT by the medical staff and diabetes patients remains insufficient; moreover, it has not garnered attention as a research topic. To investigate the ultrasound characterization of LHT, to identify the factors associated with LHT development by assessing the prevalence of LHT and compare the accuracy of clinical palpation with that of ultrasonography in LHT detection, and to further evaluate the possible impact of LHT on patients’ blood glucose fluctuations. A cross-sectional study was conducted in 120 patients with type 2 diabetes. Patients’ general information were obtained using a questionnaire, and the patients were evaluated for LHT by ultrasonography and clinical palpation of the abdomen. The patients were instructed to inject equal amounts of insulin in tissues with LHT and in normal adipose tissues (NATs) in two non-consecutive d in a selected week; the possible effect of LHT on patients’ blood glucose fluctuations was assessed using a continuous glucose monitoring system. LHT has characteristic ultrasonic signs. We found a high rate of missed LHT detection on clinical palpation compared with that on ultrasonography (P < 0.05). The duration of insulin treatment, rotation of injection sites, frequency of needle reuse, and number of insulin injections per day were the primary factors influencing the development of LHT (P < 0.05). Compared with NATs, LHT tissues showed extremely elevated amplitude of glycemic excursion, mean blood glucose levels, standard deviation of blood glucose levels and postprandial glucose excursion, and large fluctuations in blood glucose levels (P < 0.05). Ultrasonography can more accurately detect LHT than can clinical palpation. LHT development is associated with several factors and can lead to significant fluctuations in blood glucose levels; thus, sufficient attention should be paid to investigating the underlying mechanism of LHT.


Introduction
Insulin, a commonly used hypoglycemic drug, is itself a growth factor with growth-promoting effects. Insulin has a permissive effect on protein synthesis and promotes adipocyte differentiation and lipogenesis. Lipohypertrophy (LHT) can develop when insulin is repeatedly injected at the same site. LHT mainly presents as a rubbery or scar-like lesion with thickened subcutaneous tissue at the injection site [1]. At present, LHT is mostly evaluated by palpation during clinical treatment, which is a simple, convenient method that is easy to promote. However, this method lacks specificity. It cannot accurately detect early lesions and is not helpful for accurate quantitative assessment, which often leads to missed diagnoses [2]. Ultrasound is a noninvasive test that has a higher specificity for LHT compared with that of clinical palpation [3]. Recent meta-analyses have reported a mean prevalence of 38% for LHT [4], but the incidence of LHT appears to vary widely across studies, ranging from 1.9 [5] to 73.4% [6]. The wide variation in the incidence of LHT may be explained by the different diagnostic methods used in different studies (some using palpation only, others using ultrasonography). To date, the are no specific guidelines on the definitive ultrasound diagnostic criteria for LHT, and more studies are needed to verify whether clinical palpation is sufficient to detect LHT or whether color ultrasound would improve the accuracy of LHT assessment.
A previous univariate analysis study [7] showed that the formation of LHT may be related to the use of an incorrect method for insulin injection. However, further research is needed to confirm this finding. On the contrary, when insulin is injected at the LHT areas in diabetes patients, the rate of insulin absorption is decreased and delays in peak insulin levels occur, resulting in poor glycemic control and an increased incidence of hypoglycemia [8,9]. A previous multicenter clinical study in China showed that [10] patients with LHT had a significantly higher hemoglobin A1C (HbA1c) levels and required a higher insulin dose than did patients without LHT. Moreover, LHT can lead to additional insulin consumption, significantly increasing the economic burden. The relationship between glycemic control and LHT remains controversial and requires further validation.
This study aimed to explore the ultrasound representation of LHT via a cross-sectional study to identify the factors associated with the development of LHT by assessing the prevalence of LHT as detected by both clinical palpation and ultrasonography. The possible impact of LHT on patients' blood glucose fluctuations was ultimately assessed. The results of this study will help medical staff and patients to properly understand the severity of LHT, thus educating patients to maintain good injection habits during insulin use, and minimizing the development of LHT.

Study design
The participants with diabetes receiving insulin treatment who were admitted to the 900 Hospital of the Joint Logistics Team in China between April and December 2019 were enrolled. The trial was approved by the local ethics committee (2019-007) and performed in accordance with the principles of the revised Declaration of Helsinki. Written informed consent was obtained from the participants prior to their enrollment in the study. The trial was registered at ClinicalTrials.gov (registration no. ChiCTR2100048886).

Eligibility criteria
Patients aged ≥18 y at the time of screening; male or female patients; patients who met the 1999 World Health Organization diagnostic criteria for type 2 diabetes; and/or patients who had been receiving subcutaneous injections of abdominal insulin at least once a day for more than 1 y. Conversely, patients with type 1 diabetes mellitus; with a combination of acute infection, ketoacidosis, and other serious complications; with known presence of abdominal masses, such as lipomas; with a combination of inflammatory abdominal skin lesions, such as psoriasis and eczema; with known allergy to medical ultrasonic couplants; who were unable to provide their complete general information or undergo clinical physical examination; with a history of abdominal surgical treatment with significant scars; with frequent or recent severe hypoglycemia; who were pregnant; and/ or with a combination of mental illness and cognitive dysfunction, and an inability to care for themselves were excluded.

Data collection
Demographic information, results of glycemic control assessment, and the insulin injection technique used were obtained via a questionnaire administered by a physician or research nurse. The questionnaire included questions on age, sex, body mass index (BMI), HbA1c, duration of insulin treatment, total daily insulin dose, injection habits (needle length, frequency of needle reuse, and rotation), and unexplained hypoglycemic events.

Assessment of LHT
Clinical palpation assessment of LHT: Three senior nurses with a long history of diabetes education was trained to assess each patient for the presence of abdominal LHT [11]. Each patient was randomly palpated by the above three nurses, and the mean of LHT was calculated afterwards. The assessment was performed as follows: (1) the patient was placed in a lying position and instructed to fully relax their abdominal muscles, bend their knees, relax their quadriceps muscles, cross their arms over their chest, and relax their arm muscles, while the examiner took a sitting position for the examination; (2) the examiner used a light source to fully illuminate the area under examination, adjusting the angle so that any subtle elevations or depressions on the skin surface could be seen and using a marker to mark the center of the area with bumps or changes in skin color or hair distribution; (3) when palpation initially revealed a soft, elastic, subcutaneous fatty tissue that eventually changed to tough, rubbery, or inelastic tissue, the exact location of the lesion was marked using a safe skin marker.
Ultrasonography to assess LHT: Ultrasound examination of the abdominal skin tissues was performed by two senior ultrasonographers who had been practicing superficial tissue ultrasound for a significant period of time, using a multi-frequency linear probe (L8-18I, [8][9][10][11][12][13][14][15][16][17][18]. The examination was performed as follows: (1) the patient was examined in the same position as that used for clinical palpation, and (2) the ultrasound signs at the examination site and the thickness of the subcutaneous adipose tissue in the lesion area and that of the surrounding normal subcutaneous adipose tissues were recorded, and the exact location of the lesion was marked using a safe skin marker. LHT was considered to be present when the specific criteria for the ultrasound diagnosis of LHT were met [12,13]: the presence of echogenically heterogeneous nodules in the hyperplastic area with differences in echotexture from the surrounding normal tissue and interstitial edema around the hyperplastic nodules, continuous thick fascial tissue or interrupted and distorted thin connective tissue around the hyperplastic nodules, and little or absent neovascularized echogenicity in the hyperplastic nodules. Common subcutaneous masses, such as subcutaneous hemorrhage and lipoma, were excluded.

Assessment of blood glucose fluctuations
Patients were instructed to inject equal amounts of insulin subcutaneously into the LHT and normal adipose tissues (NATs) on two non-consecutive d in a selected week. To avoid the occurrence of hypoglycemia, the insulin injection dose was reduced by 10% from the original dose. In order to avoid acute hyperglycemia caused by the reduction of the insulin dose, the participants' fasting blood glucose levels should be controlled at 3.9-7.0 mmol/L the day before treatment, 2 h postprandial blood glucose should be <10 mmol/L, and approximately the same amount of activity and food intake should be maintained on the 2 d of blood glucose measurement. CGMS (Medtronic, Dublin, Ireland) was used to measure the blood glucose levels in the morning after an overnight fast, 2 h after breakfast, before lunch, 2 h after lunch, before dinner, 2 h after dinner, and at 10:00 p.m. From here, the patients' blood glucose fluctuations were evaluated. The intra-day blood glucose fluctuation index was calculated as follows: largest amplitude of glycemic excursion, which is the difference between the maximum and minimum values of blood glucose in 1 d; mean blood glucose, which is the mean of the blood glucose values in 1 d; the standard deviation of blood glucose, which is the standard deviation of the blood glucose values in 1 d; and postprandial glucose excursion, which is the mean of the difference between the blood glucose values 2 h after three meals and the corresponding pre-meal blood glucose values.

Statistical analysis
Data were analyzed using the SPSS 23.0 software (IBM Corp., Armonk, NY, USA). Descriptive data were expressed as means ± standard deviations. The Cohen's k statistic was used to determine an agreement between the clinical palpation and ultrasound assessment. The consistency in the detection between the two methods of evaluation was analyzed using the McNemar test. The differences between both groups were analyzed using a paired Student's t test for normal continuous variables and a non-parametric Wilcoxon test for non-normal data. Pearson's correlation coefficient was used to perform a correlation analysis. Results of the Pearson's correlation analysis and paired Student's t test associated with the LHT size (P < 0.05) were incorporated in the multivariate linear regression model for analysis. A P value of <0.05 was considered statistically significant.

Clinical characteristics
A total of 120 insulin-injected diabetic patients were included in this study, including 70 males and 50 females, with a mean age of 59.21 ± 11.44 years, a median insulin injection life of 6.56 (4.26) years, an average daily insulin injection of 36.08 ± 19.06 units and an average of 9.45 ± 1.85% of HbA1c. Additional information is shown in Table 1.

Characteristics of ultrasonic detection of LHT
The difference between NAT and LHT on ultrasonography is shown in Fig. 1. In normal subcutaneous tissue, the adipose tissue (hypoechoic) separated by thin connective tissue and thick myofascia ( Figure 1A). The distance between the two the thickness of the subcutaneous fat. The ultrasound features of fatty hypertrophy ( Fig. 1B-D) located in the subcutaneous tissue between the epidermal and muscular layers, and the lesions were nodular hyperechoic or interstitial edematous hypoechoic area with echogenicity different from the surrounding normal tissue.

Compared with ultrasound examination, the LHT missed rate of clinical examination
Clinical examination and ultrasonography were performed to evaluate LHT in 120 diabetic patients (as shown in Table  2). LHT was detected by clinical examination in 56 (46.6%) patients, LH was detected by ultrasound in 83 (69.1%) patients, and LHT was detected by ultrasound only in 27 (22.5%) patients. Compared to ultrasonography, the rate of LHT missed by clinical examination alone was 32.6%.
The total number of LHT detected by ultrasonography and clinical examination was further counted. In this study, a total of 144 LHT were detected by ultrasonography, and only 67 were detected by clinical examination. Compared to ultrasound examination of LHT, the rate of lesions missed in patients assessed by physical examination alone was 53.5%. When the vertical length of LHT was <5 mm, 5-10 mm and >10 mm, respectively, the leakage rate of LHT assessed by clinical examination was 94.2%, 32.9% and 10.0%, respectively. When the LHT area was <30 mm 2 , 30-60 mm 2 and >60 mm 2 , the leakage rate was 95.9%, 74.4% and 3.5%, respectively, when LHT was evaluated by clinical examination alone. LHT vertical length, LHT horizontal width and LHT area were strongly correlated with the rate of missed clinical examinations (P < 0.05) (see Table 2).

Risk factors associated with the influence of abdominal LHT
The incidence of LHT in diabetic patients who had 1, 2 and 4 subcutaneous insulin injections daily was 29.0%, 61.5% and 92.1%, respectively; the incidence of LHT in patients with <5, 5-10 and >10 years of duration of insulin treatment was 48.1%, 78.4% and 96.6%, respectively; the incidence of LHT in patients with <4, 4-8 and >8 times of needle reuse were 53.1%, 78.9% and 90.9%, respectively, and the incidence of LHT at rotational injection sites and nonrotational injection sites were 60.6% and 79.6%, respectively, with statistically significant differences (P < 0.05) (see Table 3). Gender, age, waist circumference, BMI, needle length and total daily insulin dosage had no significant effect on LHT (P > 0.05). By bringing the above statistically significant influencing factors into the binary logsitc regression model analysis, the results showed that total daily injections and duration of insulin treatment were positively associated factors affecting LHT formation [OR (95% CI) was 2.  Table 4).

Discussion
This study showed that LHT has characteristic ultrasound signs and that ultrasonography can more accurately detect LHT than does clinical palpation, and can accurately measure the LHT size and area. The years of insulin injection, rotation of injection sites, the frequency of needle reuse, and the number of daily insulin injections were the primary factors affecting the development of LHT. LHT may influence the control of patients' blood glucose, leading to increased fluctuations in blood glucose. To date, no other studies have proposed specific criteria for detecting LHT with head-to-head comparison and validation of the accuracy of clinical palpation and ultrasound. This greatly limits the use of ultrasonography in LHT. Histopathological examination showed that LHT was formed by adipocytes, which abnormally enlarged to twoto-three times the size of normal adipocytes and fibroblasts and could invade the adjacent reticular fibrous membrane and surrounding connective tissue, although often without neovascularization [14]. In this study, the following LHT ultrasound characteristics were innovatively summarized by combining previous studies in the literature [12,13]: (1) the hyperplastic area is characterized by echogenic nodules (hyperechoic or isoechoic) with a different echotexture from that of the surrounding normal tissues and interstitial edema (hypoechoic) around the hyperplastic nodules; (2) there is continuous thick fascial tissue or interrupted and distorted thin connective tissue around the hyperplastic nodules, although this may not be present in markedly obese individuals; (3) there is little or no neovascularized echogenicity within the hyperplastic nodules; and (4) the edges of the  In clinical practice, LHT is most commonly assessed via palpation. However, this method is unreliable and is associated with a high level of interclinical variability. Nurses with rigorous training in palpation techniques were able to show a 97% case detection rate, while general nurses demonstrated a missed diagnosis rate of 34% [15]. The high rate of missed clinical palpation of LHT was also suggested in the results of this study, which showed an underdiagnosis rate of 32.6% by physical examination alone compared with that by ultrasonography. This is consistent with the findings of Wang et al [16]. In this study, a total of 144 cases of LHT were detected by ultrasound, while only 67 were detected by physical examination, resulting in a 53.5% underdiagnosis rate by physical examination alone compared with that by ultrasound for LHT. In addition, when the area of the LHT was <30 mm 2 , the leakage rate of physical examination was as high as 95.9%. Moreover, as the area of hyperplasia decreased, the leakage rate of the physical examination increased. Therefore, when LHT lesions are small, physical examination alone often results in a greater degree of underdiagnosis. To our knowledge, this is the first study to explore the leakage rate of ultrasound and clinical palpation based on the area of LHT. In addition to the precise diagnostic aspects, ultrasound better detects the nature and severity of LHT compared with that detected via palpation, thus giving clinicians the opportunity to provide more detailed advice to patients [3,17]. By visualizing the LHT tissue, ultrasound images can encourage changes in the injection behavior by revealing areas of tissue   disruption, inflammation, and depth of subcutaneous tissue [18]. This has important implications in the implementation of patient education and subsequently reduces the incidence of LHT. Studies related to LHT due to faulty insulin injection techniques have been described in the literature for decades [4]. Unfortunately, most patients do not understand the severity of LHT and prefer to inject insulin in the area with LHT than in the normal injection site because of the reduced pain. Therefore, elucidation of the factors influencing the occurrence of LHT is essential to guide patients on the correct method of insulin injection. Several authors have concluded that LHT is always associated with the following factors: sex, BMI, injection device, rotation of injection sites, the injection area, needle length, insulin regimen, and daily total insulin dose [7]. Among them, the correct rotation of injection sites is the most studied and emphasized method for preventing LHT [19]. However, a number of studies also concluded that the development of LHT is independent of factors such as sex, BMI, and frequency of needle reuse [20,21]. In this study, we measured the subcutaneous fatty hyperplasia tissue using ultrasound and analyzed the main factors affecting the area. Our results showed that, in addition to the total daily injections, the rotation of injection sites, duration of insulin treatment, and frequency of needle reuse also affected the size of the hyperplastic tissue. We suggest that the primary reason for LHT caused by needle reuse is that as the number of insulin injections increases, the reused needles become deformed, barbed, and burred, increasing the resistance encountered during needle entry and extraction. This can lead to needle blockage and fracture, thus increasing the chances of infection and LHT. Another important point is that the above factors can affect the effectiveness of insulin and the accuracy of the injected dose, leading to poor glycemic control of patients, thus blindly increasing the insulin dose, resulting in the wastage of resources due to the excessive use of insulin doses, thus leading to severe hypoglycemia.
Subcutaneous insulin absorption is a key factor affecting glycemic control in insulin-treated diabetes patients. Insulin-induced LHT has been reported to impair the normal insulin uptake and affect glycemic control [22]. Almost all previous studies reported a significant reduction in insulin uptake (in some cases, clearance of radiolabeled insulin at the injection site) and glucose reduction with LHT; in some cases, patients exhibited elevated glycated hemoglobin [23][24][25]. When patients with LHT were taught to inject insulin in normal tissues, the total daily insulin dose requirements significantly reduced and glycemic control and variability improved [26,27]. In this study, patients with ultrasoundconfirmed LHT were taught to administer equal doses of insulin subcutaneously in the LHT tissues and NATs on 2 non-consecutive d of the week, while the insulin dose was reduced by 10% from the original dose to avoid hypoglycemia, in order to assess the effect of LHT on patients' blood glucose fluctuations and insulin dose. Our results showed that insulin injections at the LHT areas could significantly affect patients' glycemic control and lead to increased blood glucose fluctuations. Therefore, provision of standardized insulin injection education for diabetes patients can help them avoid injecting insulin at LHT areas, and can simultaneously achieve good glycemic management. In addition, the results of this study suggested that when the injection site was changed from fatty hypertrophic tissues to normal tissues, the insulin dose was reduced, thus avoiding the risk of hypoglycemia, effectively reducing the insulin dosage, and mitigating the medical costs.
Inevitably, this study has some limitations. First, this was a cross-sectional study that only investigated the incidence of LHT in insulin-injected diabetes patients who visited our hospital. Due to the differences in injection techniques taught at various treatment centers, it may not represent the overall incidence of insulin-injected patients. Furthermore, the small sample size of this study may have introduced errors in the analysis of the factors influencing LHT; thus, more patients need to be included for further analyses. This study was unprecedented in several ways. It provided an innovative summary of the characteristics of LHT on an ultrasound image and the first detailed comparison of the rate of missed diagnoses between ultrasound and clinical palpation based on the area of LHT.
In summary, LHT is a common comorbidity of long-term insulin therapy in diabetes patients, which not only causes damage to a patient's appearance and leads to psychological distress, but also causes additional financial burden, decreases insulin absorption, and reduces the efficacy of glycemic control. Hence, we strongly recommend the training and use of experienced health professionals to better identify LHT lesions, and the utilization of ultrasound to assist in the diagnosis when necessary. Teaching patients the proper way of injecting insulin and emphasizing the seriousness of LHT is critical to the long-term management of diabetes.

Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
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