Table 2.

Representative Animal Subject Studies

Animal model	Experimental Methods	Major Findings	Reference
Inflammation and obesity
Obese (ob/ob) mice, Diabetic (db/db) mice and Zucker (fa/fa) rats.	Tnf (gene encoding Tumor Necrosis Factor-α) mRNA transcripts were measured in adipose tissue of T2D mice and rats, and in streptozotocin-induced diabetic mice (a model of type 1 diabetes, T1D).	TNF-α mRNA transcripts were significantly increased in the adipose tissue of T2D mice and rats, but not in the adipose tissue of streptozotocin-induced T1D mice. In T2D mice, the upregulation of Tnf preceded the development of significant hyperglycemia. TNFα contributed to impaired sensitivity to insulin in the T2D models.	³
Macrophages
Obesity was induced in C57BL/6J mice by a high-fat diet (diet induced obesity).	F4/80 was used to determine the macrophage content in lean vs. obese adipose tissues in mice.	Obese adipose tissue displayed increased macrophage content compared to lean adipose tissue and TNFα was increased in obese adipose tissue with increased macrophage content.	⁴
T2D (ob/ob) obese mice	T2D (ob/ob) mice were treated with rosiglitazone for 28 days. mRNA levels of inflammatory markers were measured.	Rosiglitazone- vs. vehicle-treated mice showed decreased inflammation.	⁵
Netrin1^fl/fl LysMCre^+/− (Ntn1^Δmac) mice were studied.	Ntn1 was deleted in myeloid cells and subjected to high fat feeding. Single cell RNA-sequencing of CD45+ myeloid cells was performed.	Single-cell RNA-sequencing (RNA-seq) of CD45+ myeloid cells revealed that upon deletion of Ntn1, a significant attrition of ATMs was noted. Increased insulin sensitivity, improved lipid handling and metabolic function and decreased proinflammatory eicosanoids were observed.	¹⁵
C57BL/6J and Hif1α^fl/fl LysMCre^−/− were subjected to high fat feeding.	High-fat feeding of wild-type mice and myeloid-specific Hif1α−/− mice was used to examine the role of hypoxia-inducible factor-1α (HIF-1α) in ATMs of obese adipose tissue.	Transcriptome analysis and real-time flux measurements showed that HIF-1α did not alter the proinflammatory state of the ATMs after 8 weeks of HFD feeding.	²¹
C57BL/6J mice on a chow diet received daily intraperitoneal injection of glutamine (1 g/kg body weight) or PBS (20 mL/kg body weight) for 14 days.	qPCR analyses of Glul mRNA expression in eWAT samples was performed.	Glutamine had no effects on body weight but levels of proinflammatory genes in the eWAT were significantly lower and levels of Adipoq (adiponectin) gene were significantly higher.	²²
C57BL/6J mice were fed high-fat diet and their average daily food intake was measured for a month. When they reached 40-41g of weight, caloric restriction (CR) was initiated.	Abdominal subcutaneous, epididymal, perirenal, and mesenteric adipose tissues were analyzed for macrophage content by qPCR and immunohistochemistry for F4/80.	In the earliest days after CR to induce weight loss in obese mice, ATM content rose in the immediate time frame (approximately days 1-7 of CR), that is, leaner mice displayed higher ATM content and Lipolysis was identified to be the key process triggering increased ATM content in the adipose tissue through the process of increased macrophage migration.	²⁴
C57BL/6J mice were fed high-fat diet for 24 weeks and then switched to CR @ 70% of high-fat diet for two weeks.	VAT was subjected to single-cell RNA sequencing and stained for crown-like structures (CLS) by immunohistochemistry.	A major macrophage subpopulation in obese VAT expressed genes relevant to lipid binding and metabolism and the HFD/CR mice displayed significantly more CLS in VAT.	²⁵
C57BL/6J mice and mice with global or myeloid specific deletion of Ager (gene encoding RAGE) were fed high-fat diet.	Insulin sensitivity, the FBC content and the levels of pro and anti-inflammatory markers in the eWAT were measured.	The Ager deleted mice showed improved Insulin sensitivity, with decreased FBC content and higher levels of anti-inflammatory markers in the eWAT.	²⁷
Neutrophils
C57BL/6J mice were fed a chow or HFD from 4 to 20 weeks of age and randomized to either a sedentary group or exercise group five days/week.	Neutrophil content, neutrophil elastase activity and markers of inflammation were measured in the adipose tissue.	Exercise training reduced adipose neutrophil content, neutrophil elastase activity and markers of inflammation.	³⁶
C57BL/6J mice were fed a 60% HFD treated with Peptidyl Arginine Deiminase 4 inhibitor Cl-amidine; a compound that blocks neutrophil extracellular traps (NET) release.	Flow cytometry of adipose tissue and liver, immunohistological analysis and glucose and insulin tolerance tests were performed.	No differences in immune cell infiltration into the adipose tissue or liver were noted and there was no effect on insulin resistance.	³⁹
Mast cells
C57BL/6 mice were fed a high-fat diet for 20 weeks.	Prevalence and distribution of dead adipocytes, mast cells, macrophages (F4/80), and apoptotic cells were measured in the eWAT along with gene and/or protein expression of several adipocytokines, F4/80, mMCP6 and cleaved caspase-3.	Increased mast cell content in obese vs. lean adipose tissue was observed.	⁴⁴
WT (C57BL/6 and WBB6F1/J) and c-Kit deficient, Kit^W-sh/W-sh mice were fed a western diet for 12-13 weeks following which the mast cell stabilizer, disodium cromoglycate (DSCG) was administered.	Body weight, glucose tolerance and energy expenditure and the weight of the WAT depots were measured.	The c-Kit deficient mice on a western diet gained less weight with significant reductions in WAT depots, in parallel with improved glucose tolerance and increased energy expenditure. Administration of DSCG, attenuated body weight gain only in the WT but not the mast cell-deficient mice; mast cell content in the WAT was not affected.	⁴⁶
Kit-dependent B6.Kit^W/Wv and Kit-independent B6.Cpa3^Cre/+ mast-cell-deficient mouse strains were fed either chow diet or high-fat diet for 16 weeks.	Body weight, glucose tolerance, energy expenditure and adiposity were measured.	No differences in body mass or adiposity vs. control mice were observed and glucose and insulin intolerance were not altered in these mice.	⁴⁷
WT and Kit^w-sh/w-sh, c-Kit deficient mice at 12-weeks of age were injected with norepinephrine to stimulate thermogenesis and mast cells were reconstituted by bone marrow transplant.	Body weight, core body temperature, and indirect calorimetry were measured.	When stimulated by norepinephrine, the metabolic rate was enhanced in these mice compared to controls. Mast cell reconstitution in those mice (in the SAT) reversed these effects of norepinephrine.	⁴⁹
Eosinophils
WT (C57BL/6) and Il4-deficient male mice were subjected to chow diet or high-fat feeding for 15-24 weeks.	Body weight, glucose tolerance and insulin sensitivity were measured.	Number of eosinophils in adipose tissue in obesity was significantly reduced compared to that observed in the lean state and when mice lacked eosinophils, they were more prone to diet-induced obesity and glucose intolerance.	⁵²
C57BL/6J mice fed a high fat diet (HFD) were simultaneously given recombinant interleukin-5 (rIL5) for 8 weeks to increase adipose tissue (AT) eosinophils.	Weight gain, AT inflammation, glucose, lipid, and mixed-meal tolerance, AT insulin signaling, energy substrate utilization, energy expenditure, and white AT beiging capacity were measured.	Eosinophils were increased ~3-fold in AT of obese HFD-fed mice treated with rIL5, and thus were restored to levels observed in lean healthy mice. However, there were no significant differences in rIL5-treated mice among the comprehensive set of metabolic assays, despite the increased AT eosinophils.	⁵³
C57BL/6J mice were either fed a chow diet or a high fat diet (HFD) for 12 weeks and later switched to chow diet to induce weight loss for up to 6 weeks.	Body weight, individual tissue weights, and the levels of adipose eosinophil and macrophages were measured.	Weight loss (switching mice from HFD to low fat diet) led to increased eosinophil content in adipose tissue, in parallel with reduced ATM content, reduced inflammation, and improved tissue remodeling.	⁵⁴
Male WT (C57BL/6J) and CCR2 deficient (CCR2^−/−) mice were subjected to high-fat diet.	Insulin sensitivity, body mass, or adiposity and the number of eosinophils in different tissues were measured.	No differences in insulin sensitivity, body mass, or adiposity were observed. Increase in numbers of eosinophils in all WAT depots was observed with no differences in other organs such as liver or bone marrow.	⁵⁷
NK cells and ILC1s
WT (C57BL/6), Rag1 deficient Rag1^−/−, IFNγ deficient Ifng^−/− and Prkdc^−/−IL2rg^−/− (mice lacking mature T, B, and natural killer lymphocytes) were fed either a chow diet or high-fat diet.	In adoptive transfer experiments, adipose ILC1s were purified by flow cytometry from C57BL/6 or Ifng^−/− mice fed a HFD and transferred into Prkdc^−/−IL2rg^−/− mice on HFD. After HFD feeding for 4 weeks, recipient peripheral organs were harvested, and adoptively transferred cells were analyzed by flow cytometry.	Compared to controls not receiving the ILC1s, there were no differences in body weight but those mice receiving the ILC1s were more glucose intolerant and showed more IFN-γ-dependent interstitial fibrosis in the VAT with more CD11C+ cells and more CLS vs. PBS.	⁷³
WT (C57BL/6) and Ifng^−/− mice were fed high-fat diet for 12 weeks .	In adoptive transfer experiments, purified adipose iNK or ILC1 from HFD-fed WT and Ifng^−/− mice were transferred into Rag2^−/−Il2rg^−/− mice and further subjected to high-fat feeding for 2 weeks. Recipient peripheral organs were harvested and analyzed by flow cytometry 2 or 4 weeks after final transfer and additional HFD feeding.	In both cases, the number of “M1”-type macrophages was higher in the adipose tissue of the recipients, but especially upon the adoptive transfer of ILC1s. In both iNK or ILC1 transfers, the recipients were more glucose and insulin intolerant vs. controls, which was independent of the amount of weight gained on the HFD.	⁶⁶
WT (C57BL/6) and E4bp4^+/− mice were fed high-fat diet for 8 weeks and injected with NK cell neutralizing antibody to deplete NK cell population. To expand NK cells in vivo, mice fed chow diet or HFD for 6 weeks were intraperitoneally injected with IL-15. For the reconstitution experiments E4bp4^−/− homozygous knockout mice were used.	To reconstitute E4bp4^−/− homozygous knockout mice with NK cells, splenic NK cells were isolated from C57BL/6 WT mice that had been fed HFD for 8 weeks by negative sorting using a mouse NK cell isolation kit were used.	When NK cells were depleted with neutralizing antibodies or using E4bp4^+/− mice, improvements in insulin resistance and decreased numbers of ATM and adipose tissue inflammation were observed. When NK cells were expanded through IL15 administration or by reconstitution of NK cells into E4bp4^−/− mice, ATM numbers and adipose tissue inflammation increased, in parallel with increased insulin resistance.	⁶⁵
WT (C57BL/6) were fed high-fat diet for 8 weeks and injected with NK cell neutralizing antibody to deplete NK cell population. For other experiments Klrk1^−/− (mice lacking a major NK cell-activating receptor) and Prf1^−/− (mice lacking perforin) knockout mice were subjected to high-fat feeding.	Adipose tissues were harvested for M1/M2 macrophage markers.	There were increased numbers of Adipose tissue macrophages that were more “M2”-like in nature in both models.	⁶⁴
ILC2s
WT (C57BL/6) and IL33-deficient Il33^−/− mice were subjected to prolonged high-fat diet feeding and administered carrier-free recombinant murine IL-33.	Visceral adipose tissues were collected and the numbers of ILC2s and eosinophils were measured.	With prolonged HFD feeding, the numbers of ILC2s and eosinophils decrease in VAT/epididymal VAT.	⁷⁸
WT (C57BL/6) mice and heterozygous Red5 (Il5^Red5/+, R5) mice were maintained at thermoneutrality and administered various doses of IL-33 for over 8 days.	Subcutaneous WAT was collected at the end of the study and the levels of UCP1 expression was measured by immunoblotting technique.	IL33-mediated activation of ILC2s promoted the growth of functional beige fat in mice under thermoneutral conditions.	⁸⁰
WT (C57BL/6), IL1RL1-deficient (Il1rl1^−/−) Il5^{tdtomato-cre/+} and Il33^−/−mice were used.	IL33 was induced by either helminth infection or treatment with IL2 on these mice and the levels of ILC2s and Tregs were measured by flow cytometry.	IL-33 activates ILC2s and Treg cells in adipose tissue in homeostasis, but also after helminth infection or treatment with IL2 and ILC2-intrinsic IL33 activation was necessary for the accumulation of Treg cells.	⁸⁴
Male WT ( C57BL/6), Rag2^−/−, and Il2rg^−/− Rag2^−/− mice were fed a normal chow diet or high-fat diet (HFD) and analyzed over a period of several weeks.	Body weight, glucose and insulin tolerance tests and indirect calorimetry were measured in addition to bone marrow transplantation and gut microbiome studies.	Mice devoid of all lymphocytes (Il2rg^−/− Rag2^−/−) were resistant to HFD-induced obesity; bone marrow transplant indicated roles for ILCs in protection from diet induced obesity; the ILC2 effects on obesity resided in the small intestine.	⁸⁵
ILC3s
Male WT ( C57BL/6), Rag2^−/− and leptin deficient Lep^ob/ob (ob/ob) mice were fed either chow diet or high-fat diet.	Lung and adipose tissues were harvested and the presence of ILC3 was detected by flow cytometry.	ILC3 content increased in the adipose tissue with obesity.	⁸⁷
Wild type (WT) C57BL/6 and Winnie mice were fed either a chow diet or a high-fat diet and administered recombinant IL-22.	Body weights of mice and diarrhea and rectal bleeding scores were recorded weekly.	Winnie mice, which are predisposed to colitis, displayed impaired intestinal barriers, upon HFD. However, treatment with IL22 improved the integrity of the intestinal barrier.	⁹¹
Wild type (WT) C57BL/6 mice, were fed either a chow diet or a high-fat diet and injected with control Ig or IL22.	Glucose and insulin tolerance tests were performed and pancreatic islets were isolated and cultured overnight for rt-qPCR analysis.	In obese mice, IL22 administration modulated oxidative stress regulatory genes in pancreatic islets, suppressed ER stress and inflammation, promoted secretion of insulin and fully restored glucose homeostasis, which was followed by restitution of insulin sensitivity.	⁹²
Wild type (WT) C57BL/6, T2D (db/db) mice and IL-22r1 deficient mice fed a high-fat diet were used.	Body weights of mice were monitored and glucose and insulin tolerance tests were performed.	Mice deficient in Il22 displayed increased metabolic abnormalities upon HFD feeding; in contrast, treatment of T2D (db/db) mice or obese HFD-fed mice with IL22 resulted in reversal of hyperglycemia and insulin resistance.	⁹³
Microglia
Wild type (WT) C57BL/6 and Lep^ob/ob (ob/ob) and fed either a chow diet or a high fat diet and were treated with leptin for 5 days.	Body weights of mice were monitored and brains were collected at the end of the study for qPCR and immunohistochemistry.	HFD increased the total number of arcuate nucleus microglia in the hypothalamus with evidence of increased microglial activity.	¹⁰⁸
Wild type (WT) C57BL/6 and Global green fluorescent protein (C57BL/6-Tg (CAG-GFAP)) mice on C57BL/6 background were used.	Bone marrow from GFP mice were transplanted into WT recipients and fed either high-fat diet or chow diet following which adipose tissues and brain were extracted for qPCR and flow cytometry .	Feeding mice a HFD resulted in an approximately 30% increase in the number of GFP+ cells in the CNS when compared to the control diet; more than 80% of the GFP+ cells in the CNS bore CD45+ CD11B+ signature, indicating that these GFP+ cells were analogous to microglia and macrophages.	¹⁰⁹
Wild type (WT) C57BL/6 and Tlr4^−/− mice were used.	Mice were injected with either vehicle or TGF-β1 in the brain by intracerebroventricular surgery and fed either chow diet or a high-fat diet and their body weights were monitored and glucose and insulin tolerance tests were performed.	Brain TGF-β1 caused an increase in glucose intolerance and hepatic glucose production, which was localized in part to astrocytes using astrocyte-specific glial fibrillary acidic protein (GFAP)-driven genetic approaches.	¹²³
Wild type (WT) C57BL/6 were used.	All mice were subjected to intracerebroventricular surgery and injected with artificial or aCSF containing 4.5 μg/μL BrdU with or without AraC 15 μg/μL and maintained on either chow diet or switched to an HFD.	Feeding excess fats and excess carbohydrates to mice resulted in obesity and, in the arcuate nucleus of the hypothalamus, a rapid and significant increase in the number of microglia was observed.	¹²⁴
GFAP expressing GFAP/^CAIkkβ^+/− mice and their wild-type (WT) littermates were fed either high-fat diet or chow diet.	Body weights of mice were monitored and glucose and insulin tolerance tests were performed.	Astrocytic process plasticity and IKKβ/NF-κB are critical to the central control of blood glucose, blood pressure, and body weight in homeostasis and in chronic overnutrition states.	¹²⁶
Wild type (WT) C57BL/6 fed either control or high-fat diet containing PLX5622.	PLX5622, a CSF1R inhibitor, selectively depleted microglia from the CNS but not macrophages from the adipose tissue and the body mass and composition were measured .	Depletion of microglia protected the mice from HFD-induced body weight gain and reduced total body fat mass, but not lean mass.	¹²⁷